Phenylacetyl carbinol

Ephedrine was originally isolated as the active agent present in plant extracts used in ancient Chinese medicine for respiratoiy ailments. As long ago as 1921 the formation of optically active phenylacetyl carbinol (PAC) from benzaldehyde and pyravate by brewers yeast and cell-free yeast extracts was reported. The PAC can then be reductively animated to produce optically active L-ephedrine (Figure 4.18). L-Ephedrine is widely used in the treatment of asthma and hay fever as a bronchodilating agent and decongestant. 

ADH CD 3D HEThDP Mes nh PAC PDC PDCS.c. PDCS.u. PDCZ.w. So.5 ThDP v/S v max wt alcohol dehydrogenase circular dichroism three-dimensional 2-(hydroxyethyl)thiamine diphosphate 4-morpholineethanesulfonsaure Hill-coefficient phenylacetyl carbinol pyruvate decarboxylase PDC from Saccharomyces cerevisiae PDC from Saccharomyces uvarum PDC from Zymomonas mobilis substrate concentration necessary for half-maximal velocity thiamine diphosphate velocity vs substrate concentration maximal velocity wild-type... 

Scheme 5.13 The PDC-catalyzed synthesis of (R)-phenylacetyl-carbinol induces the stereochemistry in the industrial synthesis of (-)-ephedrine.

Examples illustrate the rapidly-growing and promising uses of cydodextrins in various operations the intensification of the conversion of hydrocortisone to prednisolone, the improvement in the yield of fermentation of lankaci-dine and podophyllotoxin, the stereoselective reduction of benzaldehyde to L-phenylacetyl carbinol, and the reduction in toxicity of vanillin to yeast, or organic toxic substances to detoxificating microorganisms. In the presence of an appropriate cyclodextrin derivative (e.g., 2,6-dimethyl-(3-cyclodextrin), lipid-like inhibitor substances are complexed. The propagation of Bordatella pertussis and the production of pertussis toxin therefore increases up to hundred-fold. Cydodextrins and their fatty acid complexes can substitute for mammalian serum in tissue cultures. 

Engel, S., Vyazmensky, M., Berkovich, D., Barak, Z., Meichuk, J., and Chipman, D. M. (2005) Column flow reactor using acetohydroxyacid synthase I from Escherichia coli as catalyst in continuous synthesis of R-phenylacetyl carbinol. BiotechnoL Bioeng. 89, 733-740. 

By 1930, biocatalysis was also integrated into multistep chemical syntheses. The manufacture of o-ephedrine was based on Neuberg s demonstration that yeast would convert benzaldehyde to phenylacetyl-carbinol [(R)-l-phenyl-l-hydroxypropan-2-one (11)] (Neuberg and Ohle, 1922). Of particular interest, because of its relation with the work of Brown and Bertrand, is the synthesis of vitamin C [L-ascorbic acid (12)]. Two... 

CO—c—o— 1 2-acetoxyacetophenone, phenylacetyl carbinol, 16-hydroxy-17-keto-steroids —CO— acetone, 6-ketocamphor, fructose... 

PDC enzymes usually prefer small aliphatic aldehydes or 2-ketoadd compounds as donors and aliphatic or aromatic aldehydes as acceptors, furnishing the corresponding acetoin and phenylacetyl carbinol derivatives [ 14). A number of PDC variants have been obtained by structure-guided directed mutagenesis with improved activity and stereoselectivity and the results have been summarized in previous reviews [6,14,48,49,56,61,62]. 

The production of (i )-phenylacetyl carbinol by fermenting yeast was one of the first industrial biotransformations and continues to be used as a first step in L-ephedrine synthesis. 

Bringer-Meier and Sahm [28] demonstrated that pyruvate decarboxylase from Sac-charomyces carlsbergensis efficiently synthesizes phenylacetyl carbinol from pyruvate and benzaldehyde, whereas pyruvate decarboxylase from Z. mobilis is unsuitable for the biotransformations due to its low affinity for benzaldehyde and remarkable substrate inhibition. 

While initial rate of phenylacetyl carbinol formation depends on the enzyme activity, final product concentration is not dependent on the enzyme activity to the same extent. 

The efficiency of the biotransformation was found to be dependent on the pj vate benzaldehyde molar ratio. The highest phenylacetyl carbinol concentration of 28.6 g/L was obtained using 200 mM benzaldehyde and a substrate-to-benzaldehyde ratio of 2.0. 

Though use of isolated purified enzymes is advantageous in that undesirable byproduct formation mediated by contaminating enzymes is avoided [37], in many industrial biotransformation processes for greater cost effectiveness the biocatalyst used is in the form of whole cells. For this reason baker s yeast, which is readily available, has attracted substantial attention from organic chemists as a catalyst for biotransformation processes. One of the first commercialized microbial biotransformation processes was baker s yeast-mediated production of (R)-phenylacetyl carbinol, where yeast pyruvate decarboxylase catalyzes acyloin formation during metabolism of sugars or pyruvate in the presence of benzaldehyde [38]. 

Netrval and Vojtisek [39] found that a number of strains belonging to Saccharomyces, Candida, and Hansenula sp. are capable of L-phenylacetyl carbinol production. The high-... 

Seely et al. [44,45] reported that mutant strains of S. cerevisiae and Candida flareri, resistant to acetaldehyde and phenylacetyl carbinol, exhibited higher productivity than the parent strains. Nevertheless, final concentrations of phenylacetyl carbinol in the reaction mixture did not exceed 10 g/L. 

Tripathi and co-workers [46] evaluated phenylacetyl carbinol biotransformation efficiency of harvested whole yeast cells, grown continuously under glucose-limited conditions at different dilution rates. They found that cells from increasing dilutions showed increasing specific rates of product formation. 

Voets and co-workers [47] carried out studies on the effect of aeration on phenylacetyl carbinol production by S. cerevisiae. They found that although pyruvate decarboxylase participates in the fermentative catabolism of sugars, yields of phenylacetyl carbinol obtained in agitated but nonaerated batch cultures were approximately 40% lower than in aerated batches. 

Agarwal with co-workers [48] mentioned that the rate of phenylacetyl carbinol formation from glucose and benzaldehyde in a 5. cerevisiae catalyzing reaction decreased gradually with time. This study, determining the optimum benzaldehyde concentration, showed that beyond a concentration of 16 mM the rate of benzaldehyde to phenylacetyl carbinol conversion decreased. The process was completely inhibited at benzaldehyde concentrations beyond 20 mM. It was suggested that benzaldehyde concentration be maintained within the range 4-16 mM for maximum rates of conversion. 

Tripathi et al. [46] cited the optimum concentration of benzaldehyde for phenylacetyl carbinol production as 10 mM. Benzaldehyde concentrations above 1.0 g/L caused cessation of growth of C. utilis [35]. A growth inhibition constant for benzaldehyde was established as 0.30 g/L. Benzaldehyde concentrations higher than 3.0 g/L caused a significant decrease in phenylacetyl carbinol productivity. The product inhibition constant for growth was estimated as 4.1 g/L phenylacetyl carbinol. 

Vojtisek and Netrval [43] studying phenylacetyl carbinol formation from sucrose, acetaldehyde, and benzaldehyde by S. carlsbergensis (variant Budvar ) detected the highest initial rate of biotransformation and the highest phenylacetyl caibinol production in the cells with Ihe lowest pyruvate decarboxylase activity. They suggested that the total amount of phenylacetyl carbinol produced would depend primarily on the actual intracellular concentration of pyruvate, i.e., biotransformation stops due to exhaustion of intracellular pyruvate, prior to inactivation of pyruvate decarboxylase. Additions of pyruvate did not influence the rate of phenylacetyl carbinol production but increased significantly the overall production of this compound. 

The initial rate of the biotransformation reaction was found to be 2.74 g phenylacetyl carbinol per liter per hour in an optimized fermentation medium, which contained peptone, 6 g/L sodium citrate, 10.5 g/L sucrose, 40-60 g/L yeast, 60 g DW/L benzal hyde, 6 g/L (increase in benzaldehyde concentration up to 8 g/L inhibited the acyloin formation almost completely) pH 4.0-5.0 [50]. The pH optimum was 4.5-5.5 when the same reaction was catalyzed by acetone powder of yeast supplemented with cofactors [51]. 

A fed-batch process used in commercial production of phenylacetyl carbinol consists of two stages ... 

Using this strategy, Wang et al. [53] reached a level of phenylacetyl carbinol accumulation up to 22 g/L. In this study the first stage of the process (cultivation of yeast) was carried out at RQ = 4-5 and the second stage (biotransformation) conducted maintaining benzaldehyde concentration of 1-2 g/L. 

Immobilization of yeast cells was shown to reduce the toxic effect of benzaldehyde because of diffusional limitations and gradients of toxic compounds that are established within the immobilizing matrix [35,54,55]. Cells of S. cerevisiae immobilized in sodium alginate beads withstand higher concentrations of benzaldehyde (up to 6 g/L) and produce more phenylacetyl carbinol [54]. Phenylacetyl carbinol production by immobilized cells was significantly higher (1.4-, 2.5-, and 7.5-fold) than that by free cells, using initial benzaldehyde concentrations of 2, 4, and 6 g/L, respectively, in fermentation medium. 

The rate and extent of biotransformation of high concentrations of benzaldehyde to phenylacetyl carbinol by inunobilized cells of S. cerevisiae were stimulated by additions of (3-cyclodextrins to fermentation medium [56]. With cyclodextrin additions of 0.5-1.5% and cumulative doses of benzaldehyde of 12 and 14 g/L, the yield of phenylacetyl carbinol obtained was about twofold higher than in the control experiment. Besides, these additions caused faster glucose consumption and benzaldehyde utilization. 

However, with immobilization of yeast cells, it is not as easy to regulate metabolism with the same efficiency as can be done with free cells. In shake flasks, cells of C. utilis, immobilized in calcium alginate beads, exhibited enhanced resistance to benzaldehyde in comparison with free cells [35]. They also produced higher levels of phenylacetyl carbinol. But in experiments with programmed feeding of benzaldehyde in a controlled bioreactor the final phenylacetyl carbinol production by immobilized cells was 15 g/L, significantly less than the 22 g/L achieved with a free cell fed-batch system. This difference in phenylacetyl carbinol productivity was attributed to the inability to regulate yeast metabolism via RQ in immobilized cells. 

Phenylacetyl carbinol production by yeast cells immobilized on carriers other than sodium alginate did not meet with great success [57]. All the six immobilized yeast systems... 

Nikolova and Ward [72,73] studied production of phenylacetyl carbinol from benz-aldehyde and pyruvate by whole-cell yeast biotransformation in two-phase systems. For the biocatalyst preparation fresh pressed commercial baker s yeast (50 g) was suspended in 50 ml 0.05 M sodium citrate buffer (pH 6.0) and lyophilized. Aliquots of 300 mg of lyophilized cells were mixed witii 1 g celite and the mixture was resuspended in 0.05 M sodium citrate buffer (pH 6.0). The suspension was lyophilized again and stored at 4°C. Scanning electron micrographs of the carrier celite and yeast cells lyophilized on celite are given in Fig. 1. Prior to use, organic solvents purchased in anhydrous form were saturated with 0,05 M sodium citrate buffer (pH 6.0). The same buffer was used as an aqueous component of the biphasic systems. 

The effect of moisture content on production of phenylacetyl carbinol by cells immobilized on celite was investigated using hexane as organic solvent (Table 3). Maximum biotransformation activity was observed with a moisture level of 10%, The effect of the solvent type on the rate of production of phenylacetyl carbinol was investigated in two-phase systems containing 10% moisture and related to log P. The results are presented in Table 4. The highest biotransformation activities were observed with hexane and hexa-decane and the lowest with chloroform and toluene. 

Table 3 Effect of Moisture Content on Phenylacetyl Carbinol Production by Whole...

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